Chromatography is basically a biophysical analytical technique which includes identification, purification and separation of components of the given mixture. This technique can be used both for qualitative as well as quantitative applications.
IUPAC definition of chromatography:
According to IUPAC system chromatography is a
separation technique in which components of separating mixture distributes
among stationary and mobile phase. There are two basic components in
chromatographic techniques
(i) Stationary phase
(ii)
Mobile Phase
(i) Stationary phase (SP):
This is the solid or fixed phase which possesses a
stick solid material adhered with a support like glass column or it may contain
a liquid layer or slurry adsorbed on a solid support through which mobile phase
containing sample runs during analysis.
(ii)
Mobile phase (MP):
It may be in liquid or gaseous phase depending upon
the type of chromatography but can never be solid. Sample or mixture to be
analyzed and separated is mixed with mobile phase which then runs through solid
phase where separation of components of the mixture takes place. Polar as well
as non-polar solvents are used as mobile phase. This phase is also known as
moving phase.
Principle of chromatography:
Chromatography is based on separation of different components of the given sample or mixture through various interaction modes. The main contributing factor in this technique is the interaction between mobile phase, stationary phase and analytes with both phases. The analyte is mixed with mobile phase and runs through solid phase where different chemical interactions and adsorption of analytes take place. Mostly among mobile and solid phases, one phase is selected as hydrophilic and the other is lipophilic, so that the separation of all types of components becomes easy. Thereby, depending on polarity stationary phases retards the flow of various components of the mixture differently and thus eliminates and separates all the components at divergent times and positions. The detector detects these components, records the signals of each component and gives the results in the form of chromatogram.
Chromatographic process:
Chromatographic process works due to difference
among distributing constant of each component of the given mixture. Thereby,
the components are separated on the basis of different chemical composition and
structures. As the mobile phase along with sample components passes through the
stationary phase, equilibrium established and each component of the sample
moves through the solid phase at different flow rate possessing different
migration rates.
Terminology used in chromatography:
1. Chromatograph
Instrument generally used to carry out
chromatographic procedure is known as chromatograph. The main components of any
chromatogram are injector, column and detector.
2. Chromatograph
The detector records the signals and plotted in the
form or chromatograph. The intensity and position of peak in a chromatograph
helps to identify the quantity and composition of the components.
3. Analyte
The component of the mixture or substance to be
separated from the sample is known as analyte.
4. Eluate
The mobile phase leaving the column after moving
through the stationary phase is known as eluate.
5. Eluent
The solvent that possesses the analyte is known as
eluent.
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